SUMMARY OF WORK This research area involves the study of embryonic stem cells prior to and during differentiation to cardiomyocytes. For these studies, we employ embryonic stem (ES) cells (R1, D3), embryonic germ cells (EG-1) and embryonic carcinoma cells (P19). Past accomplishments include establishment of efficient in vitro systems to generation of cardiomyocytes from ES cells in vitro, and the analysis of ryanodine type 2 deficient ES cells and their effects on cardiomyocytes. Selection protocols (most recently with a cardiac-restricted portion of the Na/Ca exchanger promoter) have also permitted the isolation of purified cardiomyocytes from these heterogeneous cultures. Recently, we established an in vitro model consisting of monolayer cultures of highly proliferative embryonic stem (ES) cell-derived CMs that can be employed that facilitate the analysis of cell cycle control mechanisms. This work has been submitted for publication. Separately, the research is aimed at generating cardiac-lineage specific cells to understand the role of regulatory proteins in the formation of cardiomyocytes in vitro. This includes the analysis of BMP/SMAD signalling and the inductive roles of ascorbate and suramen. An additional focus of this basic research effort is devoted to the improved viability of these cells during in vitro cultivation conditions. For this we have focused on the roles of p53 and Mdm2. Several target genes are also being modified in the stem cells to determine their role in cardiomyocyte differentiation and survival, and more recently, we have begun isolating (and targeting) cells to select sub-populations of cardiac progenitor cells that may be more appropriate for cellular based therapies. By studying the basic biology of embryonic stem cells, we hope to delineate mechanisms responsible for cardiomyocyte development and renewal, and apply these results to improve cellular based therapies that may be applicable to man.